APPLIED AND ENVIRONMENTAL , Feb. 2006, p. 1708–1715 Vol. 72, No. 2 0099-2240/06/$08.00ϩ0 doi:10.1128/AEM.72.2.1708–1715.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Diversity of within Rock Varnish in the Whipple Mountains, California† K. R. Kuhlman,1* W. G. Fusco,2 M. T. La Duc,1 L. B. Allenbach,2 C. L. Ball,2 G. M. Kuhlman,1 R. C. Anderson,1 I. K. Erickson,3 T. Stuecker,1 J. Benardini,2 J. L. Strap,2 and R. L. Crawford2 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California 911091; Environmental Biotechnology Institute, University of Idaho, Moscow, Idaho 83844-10522; and Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844-30513

Received 16 February 2005/Accepted 19 October 2005

Rock varnish from Arizona’s Whipple Mountains harbors a microbial community containing about 108 microorganisms g؊1 of varnish. Analyses of varnish phospholipid fatty acids and rRNA libraries reveal a community comprised of mostly Proteobacteria but also including Actinobacteria, eukaryota, and a few members of the . Rock varnish represents a significant niche for microbial colonization.

Rock varnish (also known as desert varnish) is a dark, thin forms very slowly at rates thought to be between Ͻ1 to about (usually 5 to 500 ␮m thick), layered veneer composed of clay 40 ␮m per 1,000 years (50), thus archeologists have been in- minerals cemented together by oxides and hydroxides of man- terested in dating the age of varnishes to place petroglyphs ganese and iron (11, 20, 56, 63, 64). Nineteenth century refer- etched into varnish by ancient cultures into their full historical ences to rock varnish include those of Humboldt (42) and context (22, 90). Unfortunately, radiocarbon dating of varnish Darwin (14). Modern observations of varnish were initiated has proven difficult, and results must be used with caution (7, with the studies of Laudermilk (49) and Engel and Sharp (25); 9, 17, 22, 62). however, despite decades of study, the nucleation and growth It has been suggested that varnish or varnish-like materials mechanisms of rock varnish remain a mystery (11, 18, 37, 44, may exist on Mars (2, 36, 44, 65). If so, varnish may be a niche 57, 58). for colonization by extraterrestrial life forms such as . Mn(II) is the soluble form of manganese that is available to Microorganisms are ubiquitous within varnishes on Earth. organisms. It is stable between pH 6 and 9. Mn(III) and Thus, the study of Earthly varnishes may lead to the proper Mn(IV) primarily form insoluble oxides and oxyhydroxides. design of experiments in coming decades for detection of life Microbial Mn(II) oxidation could thus result in the formation on other planets. For example, iron and manganese oxidation of manganese oxides as mineral phases in varnishes, as occurs by microbes cultivated from varnish has been extensively in- in other environments (23, 39). Like manganese oxidation, iron vestigated (1, 19, 26, 33, 43, 46, 47, 54, 56, 77, 78, 80, 83). Perry oxidation (95) occurs at the exterior of the cell surface. Iron et al. (59) observed a variety of amino acids in rock varnish and hydroxides are often deposited on the remains of biogenic suggested that this is evidence for an intimate association of structures (24). The extracellular deposition of ferric hydrox- bacteria with the varnish material. A large variety of bacterial ides is a way for iron-oxidizing organisms to prevent encrusta- genera have been cultivated from rock varnish. These include tion in iron oxide precipitates (88). Such precipitates might be Bacillus (43, 56), Geodermatophilus, Arthrobacter, Micrococcus, incorporated in a varnish matrix through the activities of iron- Curtobacterium, Cellulomonas (43, 48), Pedomicrobium, and a oxidizing bacteria. Metallogenium-like strain (19, 20). Eppard et al. (26) isolated Rock varnish may hold a record of the microclimate in which several actinomycete species including Geodermatophilus. Sta- it is found (7, 10, 11, 30), a hypothesis that has been questioned ley et al. (78, 79) observed microcolonial fungi on rock varnish. previously (67, 68). Some investigators suggest that rock var- nish may harbor a historical record of important environmen- Taylor-George et al. (83), Gorbushina et al. (32), and Perry tal processes such as long-term climate change (51). Bao et al. (55) have provided evidence that these fungi may be involved (7) studied preservation of atmospheric signatures in rock var- in the formation of varnish. nish and concluded that rock varnishes or other surface depos- To our knowledge, only two studies to date have investigated its may provide a record of paleoclimatic information and the microbial of varnish (26, 57). Eppard et al. sulfur biogeochemical cycles. As a deposit of submicrometer (26) used 16S rRNA gene sequencing techniques to examine layering, rock varnish may record the activity of dust storms, phylotypic characteristics of bacteria that had previously been moisture and temperature fluctuations, biological activity, and cultured from various rock varnishes. Kuhlman et al. (48) em- the occurrence of fires over thousands of years. Rock varnish ployed 16S rRNA gene sequencing to identify several UV light-resistant bacteria isolated from rock varnish obtained in the Whipple Mountains of the U.S. Mojave Desert. These * Corresponding author. Present address: Planetary Science Insti- strains included representatives of the genera Geodermatophi- tute, 1700 East Fort Lowell Rd., Suite 106, Tucson, AZ 85719. Phone: lus, Arthrobacter, Curtobacterium, and Cellulomonas. There are (520) 622-6300. Fax: (520) 622-8060. E-mail: [email protected]. † Supplemental material for this article may be found at http://aem several reports of “microcolonial” fungi living within rock var- .asm.org/. nish or on the surfaces of rocks in hot, dry deserts (45, 55, 56,

1708 VOL. 72, 2006 DIVERSITY OF MICROORGANISMS WITHIN ROCK VARNISH 1709

78, 79, 83). The first scanning electron microscope images of homogeneous, and incubated at room temperature for 30 min. such microcolonial fungi were reported from samples collected The samples were stained with 60 ␮lmlϪ1 of a stock 4Ј,6Ј- in the Sonoran Desert in 1978 (55). The fungi observed were diamidino-2-phenylindole (DAPI) or acridine orange (50 ␮g thought to be representatives of the ascomycetes. It has been mlϪ1) solution. DAPI-stained samples were incubated at room suggested that these fungi are the predominant biological temperature for 30 min in the dark before filtration. The most forms observed on rock varnish coatings (32, 55, 60, 79, 80) and appropriate volume for analysis was determined to be 500 ␮lof may be involved in the formation of varnish (83). the 10Ϫ3 dilution. This dilution contained Ͼ25 but Ͻ250 cells Most organisms in nature are refractory to cultivation (4, 8, per field and had a low enough mineral content to both count 85, 89); therefore, many organisms that occupy the varnish cells in about one microscopic plane and not have cells ob- habitat remain to be discovered. We report here the charac- scured by the varnish minerals. Samples were filtered onto terization of microbial rock varnish communities from the 25-mm Millipore Isopore 0.22-␮m pore-size black polycarbon- Whipple Mountains of the American Sonoran Desert via prep- ate filters (Millipore, Billerica, Mass.) with Whatman 25-mm aration of 16S and 18S rRNA gene clone libraries. These GF/F filters used for support. Fluorescing cells were counted analyses are leading us to a greater understanding of the mi- on a Zeiss Research epifluorescence microscope equipped crobial diversity within these communities and their relevance with an Osram xenon short arc photo optic lamp XBO 75W to the possible occurrence of microbial life on other planets, and Chroma no. 31000 filter set for DAPI/Hoechst/AMCA such as Mars, where life forms face exposure to high fluxes of (Zeiss, Inc., Thornwood, N.Y.). The mean number of fields damaging UV light (13) and extremes of temperature and counted per sample (n ϭ 15) was 57.16. The standard deviation desiccation, such as are seen in locations on Earth where rock per sample was 7.48 fields. The average DAPI direct count of varnishes form. Microorganisms such as those that live or sur- the rock varnish was 9.0 ϫ 107 cells gϪ1 (standard deviation ϭ vive in varnish may also be potential forward contaminants on 1.2 ϫ 107). There was no difference between DAPI and acri- spacecraft structures (73). dine orange direct counts. Varnish samples were collected from alluvial fan deposits PLFA analyses were carried out by Microbial Insights, Inc. surrounding the Whipple Mountains, California. The Whipple (Rockford, Tenn.). Total lipids were extracted (91) and the Mountains lie west of Parker, Arizona, along the eastern polar lipids separated by column chromatography (35). The boundary of the Mojave Desert. Previous studies conducted at polar lipid fatty acids were derivatized to fatty acid methyl the site demonstrated relatively thick varnishes (5 to 100 ␮m esters, which were quantified using gas chromatography (69). thick) on various rock types (5). Varnished rocks and sur- Fatty acid chemical structures were verified by chromatogra- rounding soil samples were collected in February 2003 when phy/mass spectrometry and equivalent chain length analysis. the ground was still wet from winter rains. Only clasts with PLFA are essential components of the membranes of all cells thick coatings (greater than 50 ␮m) on relatively large, flat except those of the Archaea, so their profiles allow for exam- surfaces were collected. It was critical that the samples be ination of most of the important members of many microbial collected as aseptically as possible. Sample purity can never be communities. Since phospholipids break down rapidly upon completely verified, since it is very difficult to characterize cell death in environments examined thus far (91, 92), PLFA contamination associated with local wildlife. However, con- analysis should be also an accurate method for determining the taminant potential was monitored during phylogenetic inves- amount of viable microbial in an environment (92). tigations by preparing control DNA libraries from soil adjacent The sum of the PLFA expressed in pmol is proportional to the to the point of collection of varnished rock. Varnished rocks number of cells, sensu lato. The proportion used here was were approached from the downwind direction, photographed 20,000 cells pmolϪ1 total PLFA (Microbial Insights, Inc., in situ, picked up at arm’s length using sterile gloves, and Rockford, TN). The number of cells within the microbial com- placed within sterile Whirl-pak bags large enough to hold en- munity from a sample of rock varnish was thus determined tire rocks, and bags were sealed. Loose dirt on the undersides from its PLFA content (31.253 pmol gϪ1 [dry weight]). The of the varnished rocks was brushed off in the field. The bags microbial content of the varnish was ϳ108 cells gϪ1 (dry were then wrapped in protective material to prevent damage weight), in excellent agreement with the direct microscopic and subsequent contamination. The varnish was harvested counts of 9.0 ϫ 107 Ϯ 1.2 ϫ 107 cells gϪ1. The PLFA profile of from the host rock in a laminar flow bench. A Dremel grinding biomass within the varnish indicated a relatively simple com- tool with flame-sterilized coarse bit was used to grind the munity structure primarily composed of monoenoic PLFA, varnish from the host rock into a sterile container. This ap- indicative of Proteobacteria with smaller but significant popu- proach minimized possibilities for atmospheric contamination lations of Actinobacteria and Eukarya (Table 1). of the varnish and ensured that the majority of bacteria re- Three rRNA gene libraries from the Whipple Mountains moved came from within the varnish matrix. rock varnish community DNA and two control libraries from Microbial biomass for enumeration and phospholipid fatty soil lacking visible varnish adjacent to the varnished rock were acid (PLFA) analysis was obtained from powdered rock var- generated using DNA isolated directly from 0.5 g ground var- nish (0.1 g) obtained by aseptically grinding the varnish from nish and 0.5 g adjacent, unvarnished soils with an Ultraclean the rock surface and then adding the varnish to 1 ml of sterile, soil DNA kit (Mo Bio, Solana Beach, Calif.). We were careful double-distilled water (18 M⍀ MilliQ water) in a 1.5-ml sterile to avoid collecting rock fragments with our soil samples. Var- microcentrifuge tube. Serial 1:10 dilutions were made, giving a nish 16S rRNA gene libraries were prepared for Bacteria and range of dilutions from 10Ϫ1 to 10Ϫ3. The dilution samples Archaea, and an 18S rRNA gene library for was prepared for were fixed with 2% ice-cold, high-performance liquid chroma- Eukarya. for 16S and 18S rRNA genes were PCR am- tography-grade methanol, vortexed until the sample appeared plified from purified DNA using primers specific for Bacteria 1710 KUHLMAN ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 1. PLFA analysis of Whipple Mountain rock varnisha rRNA gene sequences are shown in Fig. 1 and 2, respectively. Parameter (unit) Value The closest database relatives of all sequences were chosen as reference sequences for phylogenetic analysis based on Biomass (pmol PLFA gϪ1 dry weight) ...... 31,253 Cells (gϪ1 dry weight)...... 6.25 ϫ 108 BLAST (3) comparisons. The 16S and 18S rRNA gene se- % Gram negatives and firmicutes (terminally branched, quences were aligned using MegAlign (DNASTAR, Inc., Mad- saturated PLFA)...... 2.4 ison, Wis.). The model for the 16S data set (GTRϩIϩ⌫) was % Proteobacteria (monoenoic PLFA)...... 81.8 selected based on relative goodness of fit. The relative good- % Actinomycetes and sulfate-reducing bacteria (mid- ness of fit of 16 alternative models of sequence was chain branched, saturated PLFA)...... 2.3 % General (normal saturated PLFA) ...... 13.0 compared using the likelihood ratio test (81). The model for % Eukaryotes (polyenoic PLFA) ...... 0.5 the 18S data set (TrNefϩIϩ⌫) was selected using DT-ModSel (53). Maximum likelihood searches were conducted using a PLFA signatures of anaerobic metal reducers were not observed (branched monosaturated PLFA); physiological status is nonstressed (cyclopropyl acid/cis PAUP* (version 4.0b10) (82) with stepwise addition (10 ran- acid ratios and trans/cis ratios were both 0.00). dom sequence additions) and tree bisection-reconnection branch swapping. Nodal support was estimated using bootstrap analysis (100 replicates) under the appropriate model. Poste- (338f/907r) (84), Archaea (A21f/A958r) (29), and Eukarya rior probabilities (107 generations, sampling every 100 gener- (NS3/NS8) (94). Clone libraries were prepared using a TA ations) were determined under the GTRϩIϩ⌫ model of se- cloning kit (Invitrogen, Carlsbad, Calif.); 100 to 200 clones quence evolution using MrBayes (MRBAYES 3.0b4) (40). were prepared for each library. To better assess the diversity of Multiple independent runs were performed, and the parame- the varnish community with the available resources, analysis of ter values were visually inspected to ensure convergence. The more partial-length sequences was favored over analysis of diversity of the ribotypes in each clone library were compared fewer, full-length sequences. Partial sequences are a reason- by rarefaction analysis calculated in DOTUR (72). able compromise for the assessment of diversity which does not PLFA analysis (Table 1) provides indirect evidence that require precise taxonomic placement (12, 76). Each clone li- these bacteria are physiologically active (see below). It is pos- brary was further classified by placing clones into restriction sible that products produced by varnish bacteria (e.g., pig- fragment length polymorphism (RFLP) restriction group pat- ments) may play important roles in varnish morphogenesis. We terns by digesting the DNA of individual clones with HhaI did not observe organisms known to be involved in metal followed by electrophoresis on a 2% SeaPlaque agarose gel. oxidation. Since rarefaction analysis (see Fig. S1 in the supple- Banding patterns were compared (34) by visual inspection of mental material) for the bacterial varnish clone libraries failed restriction digests of each clone, and a representative clone to reach saturation, it is possible that ribotypes related to metal from each distinct RFLP group was sequenced. Sequencing oxidizers were simply not sampled from the larger, diverse was performed using an ABI Prism 3100 automated DNA varnish community. It is possible that ribotypes related to sequencer (Applied Biosystems, Foster City, Calif.). Individual known metal oxidizers may have been found if the total diver- sequencing reactions were performed using the T7 and T3 sity within the varnish had been assessed. Within the 16S primers, and a contiguous sequence was generated using rRNA gene libraries of the uncultivated microbial rock varnish ContigExpress software (Vector NTI-Informax; Invitrogen community, we observed a number of sequences related to Corp., Carlsbad, Calif.). Clone sequences were evaluated using known environmental strains but not to known metal oxidizers. the Chimera Check program implemented in the Ribosomal rRNA gene analyses are not sufficient in themselves to deter- Database Project (52). Two chimeras were found in both the mine which organisms are an integral part of the rock varnish Bacteria and Eukarya sequence libraries, while one chimera community over geologic time scales but do give an indication was found in the surrounding soil bacterial library as well as the of the phylogenetic groups that can occupy this niche at a varnish-associated Archaea library. These chimeras were ex- particular moment in time. We previously confirmed by pure cluded from further analysis. No chimeras were found in the culture techniques that viable radiation-tolerant bacteria are surrounding soil Archaea library. Nonchimeric sequences were present in Whipple Mountains rock varnish. We isolated five matched by a standard BLAST search (3) within the NCBI UV-resistant bacterial cultures on tryptic soy agar plates after GenBank database to determine closest matches. Sequences exposure to UV irradiation treatment. All of these strains were were submitted to GenBank for rock varnish Bacteria, Archaea, found to be related to known strains of Actinobacteria lineages and Eukarya and nonvarnished soil Bacteria and Archaea (see (48). However, since most members of the varnish microbial “ sequence accession numbers” below). community observed by molecular techniques were not culti- Nine distinct RFLP groups obtained from 107 bacterial vated (Fig. 1 and 2; see Tables S2 and S3 in the supplemental clones, 13 groups from 57 Eukarya clones, and two groups from material), it is possible that these organisms are indeed able to 76 Archaea clones were analyzed. Rarefaction analysis (see oxidize iron and/or manganese. We cannot know for certain Fig. S1 in the supplemental material) of the Archaea and the unless the observed strains are eventually cultivated for phys- surrounding soil Archaea libraries reached saturation, indicat- iological studies or direct in situ techniques are developed to ing these two communities were well sampled and of low di- observe microbe-associated metal oxidation within the intact versity. In contrast, rarefaction curves for the Bacteria, Eu- varnish matrix. karya, and surrounding soil Bacteria did not yet reach a Within the 16S rRNA gene libraries of the uncultivated plateau, indicating these populations were even more diverse microbial community of Whipple Mountains rock varnish, we than our initial phylogenetic analyses suggest. observed a number of sequences related to known environ- The phylogenetic relationships of bacterial and eukaryotic mental strains. Among the most common sequences observed VOL. 72, 2006 DIVERSITY OF MICROORGANISMS WITHIN ROCK VARNISH 1711

FIG. 1. Phylogenetic tree showing the relationships of bacterial 16S rRNA gene sequences to one another and to known microbial groups using 1,573 aligned characters. Nodal support values with bootstrap support (100 replicates) and posterior probabilities (107 generations), respectively, ϭϪ ϩ ϩ⌫ ϭ ␣ϭ are listed next to the branches of the unrooted maximum likelihood tree (ln L 4,404.18186 [TrNef I model]; pinv 0.512289; 0.599910). Line length, 0.1 substitutions/site; DRV, desert rock varnish isolate. Accession numbers are shown in boldface type or in parentheses. were those closely related to the genus Rubrobacter (clones were amplified by PCR from rock varnish obtained near So- DRV B1, DRV B9, and DRV B35) (see Table S2 in the corro, New Mexico. Rubrobacter species are of actinobacterial supplemental material). Van de Kamp et al. (87) reported in lineage and are known to inhabit masonry and lime wall paint- an abstract that actinobacterial 16S rRNA gene sequences ings, where they cause a rosy discoloration (71). Diverse, yet- 1712 KUHLMAN ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 2. Phylogenetic tree showing the relationships of eukaryotic 18S rRNA gene sequences to one another and to known microbial groups using 1,201 aligned characters. Nodal support values with bootstrap support (100 replicates) and posterior probabilities (107 generations), ϭϪ ϩ ϩ⌫ ϭ respectively, are listed next to the branches of the unrooted maximum likelihood tree (ln L 4,404.18186 [TrNef I model]; pinv 0.512289; ␣ϭ0.599910). Line length, 0.1 substitutions/site; DRV, desert rock varnish isolate. Accession numbers are shown in boldface type or in parentheses.

to-be-cultured members of the Rubrobacter subdivision are munity. This genus inhabits environments such as the deep also widespread in Australian arid soils (38). The sequences of subsurface (6) and has recently been observed within an en- uncultivated bacteria we observed are related to Rubrobacter dolithic community in Antarctica where it was found within radiotolerans and Rubrobacter taiwanensis, bacteria known for translucent gypsum crusts on the surface of ice-free sandstone their exceptional resistance to gamma radiation (28). One rock boulders (41). This, like rock varnish, represents an environ- varnish-inhabiting genus observed in clone DRV B85 was re- ment exposed to high levels of UV irradiation. The genus lated to Chondromyces or Polyangium (both 96% similarities). Sphingomonas contains many representatives that are able to These genera are in the lineage of Myxococcales (75), which degrade compounds such as polynuclear aromatic or haloge- contains many members known to inhabit extreme environ- nated molecules that might be present at low concentrations in ments (15) and to make bioactive substances (66). the atmosphere (93). A likely representative of the genus Sphingomonas (clone Clone DRV B27 is a potential member of the genus Rho- DRV B19) was observed in the uncultivated rock varnish com- dopila (93% similarity), an obligatory aerobic, bacteriochloro- VOL. 72, 2006 DIVERSITY OF MICROORGANISMS WITHIN ROCK VARNISH 1713 phyll a-containing bacterium genus. While 93% similarity is and also implies that other photosynthetic forms (e.g., bacte- not sufficient for unequivocal identification at the genus level, riochlorophyll a-containing bacteria such as Rhodopila, as rep- where 95% is the commonly accepted, albeit contentious, stan- resented by clone DRV 27) might be found in varnish-based dard (4, 27, 70, 72), this GenBank match hints at a possible ecosystems. This hypothesis merits testing at a variety of var- mechanism of bacterial survival in rock varnish. The environ- nish sites within both hot and cold deserts. ment of rock varnish would obviously be conducive to the Sequence analyses of representative clones of RFLP groups development of photosynthetic life forms such as Rhodopila. within an Archaea rRNA gene library (see Table S1 in the As is normal for this type of investigation, several sequences supplemental material) revealed only two RFLP types with 76 (Fig. 1) (clones DRV B9, DRV B11, DRV B19, DRV B27, and clones analyzed. Both types were related to previously ob- DRV B35) showed similarities to uncultured bacteria. This is served uncultivated Archaea. Rarefaction curves obtained from confirmation that, as in other environments, many or perhaps the archaeal clone library reached saturation (see Fig. S1 in the most of the bacteria in rock varnish have never been cultured. supplemental material). Thus, Archaea appear to be present in To our knowledge, PLFA analyses of rock varnish microbial low diversity in Whipple Mountains rock varnish. communities have not been performed previously. Lipid anal- Eukaryota observed by preparation of an 18S rRNA gene yses are a very useful supplement to DNA analyses when library from rock varnish (Fig. 2; see Table S2 in the supple- characterizing a complex microbial community such as the one mental material) were dominated by RFLP groups of fungal studied here (21, 61). PLFA analysis of the rock varnish mi- representatives of the ascomycota and included representa- crobial community (Table 1) supports the conclusions made tives of the genera Phoma, Plicaria, Ascobolus, Alternaria, Sar- from our rRNA gene analyses. The PLFA profiles of biomass cosphaera, and Rhizina. One RFLP group representative most within the varnish indicate a relatively simple community struc- closely aligned with the basidiomycota (the genus Athelia in the ture primarily composed of monoenoic PLFA, indicative of family Corticiaceae; clone DRV E112). The dominance of as- Proteobacteria. The PLFA profiles also show the presence of a comycota in our library is in accord with the microscopic ob- smaller but significant population of Actinobacteria (mid-chain servations of Perry (55), who characterized by scanning elec- Proteobacteria Actinobacteria branched saturated PLFA). and tron microscopy a microcolonial from varnish also are of particular interest in that they represent organisms that collected in the Sonoran Desert. Staley and colleagues identi- have the ability to utilize a wide range of carbon sources and fied this organism as an ascomycete within the family Demati- adapt quickly to environmental change, such as would be the aceae (78, 79). The 18S rRNA gene sequences we observed situation in the rock varnish environment. PLFA also indicated were not of this family but fell into families such as Pleospo- the presence of eukaryota (e.g., fungi), and this was confirmed raceae (clone DRV E78), Rhizinaceae (clone DRV E102), Pe- by 18S rRNA gene fingerprinting (Fig. 2; see Table S2 in the zizaceae (clones DRV E8 and DRV E36), and Ascobolaceae supplemental material). (clone DRV E56). The membranes of microorganisms adapt to the changing Sequence analyses of representative clones of bacterial and conditions of an environment, particularly under stressful con- archaeal rRNA gene libraries of nonvarnished soil (see Table ditions. These changes are reflected in the profiles of PLFA, S3 in the supplemental material) revealed mostly lineages dif- with a change of cis fatty acids toward more trans-configured acids (35). Also, the Proteobacteria respond to starvation by ferent from those seen in rock varnish libraries. Only two making cyclopropyl (35) or mid-chain branched fatty acids nonvarnish clones overlapped. These were a Rubrobacter ra- (86). The “physiological status” of a microbial community can diotolerans-like sequence and a Sphingomonas-like sequence. thus be assessed by dividing the amount of the stress-induced Both of these groups could reasonably be expected to inhabit fatty acids by the amount of their precursors (Microbial In- both soil and varnish habitats. sights, Rockford, Tenn.). Biomarker ratios calculated for the The analyses reported here are not quantitative, so little can rock varnish community harvested during the winter (February be said of the overall importance of individual RFLP groups or 2003) season (wet at the time of sampling) were indicative of a phylogenetic taxa observed to the overall community compo- community not experiencing starvation or unusual stress. Cy- sition. In this regard, it is of interest that sequences similar to clopropyl acid/cis acid ratios and trans/cis ratios were both 0.00. those of UV-resistant pure cultures isolated from the same Other microbial populations inhabit the surface environ- Whipple Mountains rock varnish used in work reported here ment of rocks in what is termed an “endolithic” lifestyle. En- (e.g., Geodermatophilus, Arthrobacter, Curtobacterium, and Cel- doliths are found within the rock matrix just below the surface lulomonas) (48) were not observed in varnish 16S rRNA gene and occur in both hot and cold deserts (31). Endolithic com- libraries. This indicates that these strains, though present, munities are invariably colonized by cyanobacterial represen- probably do not dominate the community, a result that is tatives of the Bacteria that, through photosynthesis, provide the supported by rarefaction analysis that revealed that the varnish fundamental source of carbon and energy that supports the contains a higher diversity of both Bacteria and Eukarya than endolithic community (16, 74). It is important while sampling was sampled in this study. It should be possible in future work rock varnish to be certain that endolithic communities are not to design PCR primers targeting specific RFLP groups (e.g., also collected inadvertently. We observed no 16S rRNA gene the Rubrobacter or ascomycota groups) and employ these using sequences indicative of in our samples of var- quantitative PCR techniques, such as real-time PCR, to exam- nish, even though we employed conventional primers targeting ine the quantitative compositions of rock varnish microbial conserved regions of 16S rRNA genes that should have de- communities. In addition, quantitative real-time PCR using tected cyanobacterial sequences. This is support that we sam- primers specific for enzymes known to be involved in metal pled true varnish communities and not endolithic communities oxidation may shed light on the roles that the microbial com- 1714 KUHLMAN ET AL. APPL.ENVIRON.MICROBIOL. munities found within rock varnish play with respect to the 15. Dawid, W. 2000. Biology and global distribution of myxobacteria in soils. formation and maintenance of the varnish. FEMS Microbiol. Rev. 24:403–427. 16. de los Rios, A., J. Wierzchos, L. G. Sancho, and C. Ascaso. 2003. Acid Nucleotide sequence accession numbers. Sequences were microenvironments in microbial biofilms of antarctic endolithic microeco- submitted to GenBank under the following accession numbers: systems. Environ. Microbiol. 5:231–237. 17. Dorn, R. I. 1998. Ambiguities in direct dating of rock surfaces using radio- rock varnish Bacteria, AY923078 to AY923086; rock varnish carbon measurements—response. Science 280:2135–2139. Archaea, AY923076 and AY923077; rock varnish Eukarya, 18. Dorn, R. I. 1998. Rock coatings. Dev. Earth Surf. Process 6:444. AY923087 to AY923102; nonvarnished soil Bacteria and Ar- 19. Dorn, R. I., and T. M. Oberlander. 1981. Microbial origin of desert varnish. Science 213:1245–1247. chaea, AY923105 and AY92310. 20. Dorn, R. I., and T. M. Oberlander. 1982. Rock varnish. Prog. Geogr. 6:317– 367. The research described in this publication was carried out at the Jet 21. Dowling, N. J. E., F. Widdel, and D. C. White. 1986. Phospholipid ester- Propulsion Laboratory, California Institute of Technology, under a linked fatty add biomarkers of acetate-oxidizing sulfate reducers and other contract with the National Aeronautics and Space Administration and sulfide forming bacteria. J. Gen. Microbiol. 132:1815–1825. 22. Dragovich, D. 2000. Rock engraving chronologies and accelerator mass spec- at the Environmental Biotechnology Institute at the University of trometry radiocarbon age of desert varnish. J. Archaeol. Sci. 27:871–876. Idaho. This work was supported by an award from the Director’s 23. Ehrlich, H. L. 1996. How microbes influence mineral growth and dissolution. Research and Development Fund at JPL (JPL CREI contract number Chem. Geol. 132:5–9. NAS 7-1260). 24. Emerson, D. 2000. Microbial oxidation of Fe(II) at circumneutral pH, p. We thank Cornelia Sawatzky for editorial assistance. 31–52. In D. R. Lovley (ed.), Environmental microbe-metal interactions. ASM Press, Washington, D.C. 25. Engel, C. G., and R. S. Sharp. 1958. Chemical data on desert varnish. Geol. ADDENDUM IN PROOF Bull. 69:487–518. 26. Eppard, M., W. E. Krumbein, C. Koch, E. Rhiel, J. T. Staley, and E. Since we submitted our paper another article that is relevant Stackebrandt. 1996. Morphological, physiological, and molecular character- to work reported here was published (R. T. Schelble, G. D. ization of actinomycetes isolated from dry soil, rocks, and monument sur- faces. Arch. Microbiol. 166:12–22. McDonald, J. A. Hall, and K. H. Nealson, Geomicrobiol. J. 27. Everett, K., R. Bush, and A. Andersen. 1999. Emended description of the 22:353–360, 2005). 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